There have been many controversies in human tumor immunology over the last decade, some investigators feeling that they have strong evidence for specific cell-mediated and humoral immune responses to human tumor associated antigens, and other investigators questioning the specificity of these responses. Bladder carcinomas belong to those most studied in this respect. Work, which we have conductd with support from this grant has led to the development of better serological assays for characterization of immune responses to tumor antigens. These include a modified version of the original microcytotoxicity test, which is rapid and totally objective (counting of cells being done electronically with an image analyzer), an antibody binding assay based on the use of radioiodinated protein A, and a modified radioimmunoprecipitation test, and we have used these assays with mouse sarcomas (as models), and outlined a number of experiments in our ongoing grant, in which they are to be used to characterize immune responses to bladder tumors in mice and in human patients. Since our grant application was written almost a year ago, there have been two important developments in our laboratory, which lead us to ask for supplemental funding. We have been able to adopt the Milstein hybridoma technique to raise monoclonal antibodies to mouse sarcomas, and recently also to antigens associated with mouse bladder carcinomas and a human melanoma line. We have also been able to establish several lines of typical mouse bladder carcinoma cells, which grow well in culture. This has made it possible to vastly expand our planned experiments, trying to find the specificity, and the chemical nature, of antigens associated with bladder carcinomas in mice and man. Monoclonal antibodies will be raised to such antigens (using heteroimmunization procedures in the experiments with human material), and these antibodies will be used to make immunoadsorbents for purifying the tumor antigens. They will also be employed for extensive tests of antibody specificity.